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Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

Chen H, Babino D, Schoenbichler SA, Arkhipova V, Töchterle S, Martin F, Huck CW, von Lintig J, Meyer D - PLoS ONE (2015)

Bottom Line: We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production.Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization.HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology/CMBI, University of Innsbruck, Technikerstrasse 25, 6020, Innsbruck, Austria.

ABSTRACT
Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

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Rbp7a-GFP and Nmnat1-Rbp7a-GFP are enriched in microsome preparation.[A] Experimental overview indicating the encoded proteins, concentrations of injected mRNA (2nl/embryo) and data quantification of Western-Blot shown in [B]. Relative signal intensities always relate to H2B-GFP signals (*). Signal ratios correspond to the indicated quotient of t relative signal intensities. All mRNA were co-injected with 50ng/μl nucRFP mRNA (**). [B] Anti-GFP based Western-Blot analyses of whole embryo extracts (~1,7 embryos/lane), microsome preparation (~7,5 embryos/lane) and nuclear extracts (~1,5 embryos/lane). [C] Diagram showing indicated relative signal ratios shown in [A].
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pone.0143825.g006: Rbp7a-GFP and Nmnat1-Rbp7a-GFP are enriched in microsome preparation.[A] Experimental overview indicating the encoded proteins, concentrations of injected mRNA (2nl/embryo) and data quantification of Western-Blot shown in [B]. Relative signal intensities always relate to H2B-GFP signals (*). Signal ratios correspond to the indicated quotient of t relative signal intensities. All mRNA were co-injected with 50ng/μl nucRFP mRNA (**). [B] Anti-GFP based Western-Blot analyses of whole embryo extracts (~1,7 embryos/lane), microsome preparation (~7,5 embryos/lane) and nuclear extracts (~1,5 embryos/lane). [C] Diagram showing indicated relative signal ratios shown in [A].

Mentions: The mild increase in overall NAD+ levels seen in nmnat1-rbp7a injected embryos is unlikely to have a major impact on retinoid metabolism. However, there is evidence for organelle specific, non-redundant activities of Nmnat proteins [42–45]. In humans, the three NMNAT proteins have been shown to exert slightly different enzymatic activities at different subcellular localization [42, 43]. In particular, the use of tagged proteins revealed organelle specific targeting of Nmnat1 to the nucleus, of Nmnat2 to the Golgi complex and of Nmnat3 to the mitochondria. Therefore, we hypothesized that Nmnat1 and Nmnat1-Rbp7a also might differ in their sub-cellular localizations. This notion was supported by protein sequence based prediction analysis (PSORT server) that suggested a nuclear bias for Nmnat1 (56,5%) and a dominant cytoplasmic bias for Nmnat1-Rbp7a (60,9%) and Rbp7a (52,2%, Fig 5A). To verify these predictions, fusion constructs of full-length proteins tagged with C-terminal eGFP were generated and expressed in HEK cells (Fig 5B) as well as in zebrafish embryos (Fig 5C). For the zebrafish experiments, embryos at the 1–2 cell stage were injected with a mixture of two mRNAs, one encoding the GFP-tagged protein and the second encoding a nuclear specific RFP-variant (H2B-RFP) as an injection control. As described for the human fusion protein, zebrafish Nmnat1-GFP was strictly nuclear in both systems (Fig 5B and 5C compare with H2B-GFP). In contrast, Rbp7a-GFP showed the predicted cytoplasmic distribution in HEK cells and an additional slight nuclear enrichment in the early zebrafish embryo. Importantly, also the Nmnat1-Rbp7a-GFP fusion isoform was detected in both the nucleus and the cytoplasm. The data suggest an Rbp7a-dependend expansion of the NAD+ catalyzing activity into the cytoplasm. As the majority of cellular retinal-generating capacity resides in the ER, we speculated that Nmnat1-Rbp7a might function as a ER-specific NAD+ source [8]. To test this option, different cellular fractions prepared from embryos injected with mRNA encoding the different GFP-tagged proteins were analyzed by Western-blots using an GFP-antibody (summarized in Fig 6A, for details see Material and Methods). Importantly, signals for Rbp7a-GFP (lane 3) and Nmnat1-Rbp7a-GFP (lane 5) as compared to Nmnat (lane 4) were much stronger in the ER-containing microsome fractions as compared to whole embryo extracts and the nuclear/debris fractions (termed ‘nuclear fraction’, Fig 6B). Signal quantification revealed that in whole embryo extract the protein levels for Rbp7a-GFP and Nmnat1-Rbp7a-GFP were similar to that of H2B-GFP and roughly 6–8 times lower than those of Nmnat1-GFP (Fig 6A and 6B). In contrast, in the microsome preparation Rbp7a-GFP and Nmnat1-Rbp7a-GFP level were more than 8 times higher than that of H2B-GFP and only 1,5–1,6 lower than that of Nmnat1-GFP. The data correspond to a microsome specific enrichment of Rbp7a-GFP and Nmnat1-Rbp7a-GFP over H2B-GFP by factors of 8,6 and 6,2, respectively (Fig 6A and 6C). This suggests a similar association of Rbp7a and Nmnat1-Rbp7a with microsomal structures, presumably with the ER.


Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

Chen H, Babino D, Schoenbichler SA, Arkhipova V, Töchterle S, Martin F, Huck CW, von Lintig J, Meyer D - PLoS ONE (2015)

Rbp7a-GFP and Nmnat1-Rbp7a-GFP are enriched in microsome preparation.[A] Experimental overview indicating the encoded proteins, concentrations of injected mRNA (2nl/embryo) and data quantification of Western-Blot shown in [B]. Relative signal intensities always relate to H2B-GFP signals (*). Signal ratios correspond to the indicated quotient of t relative signal intensities. All mRNA were co-injected with 50ng/μl nucRFP mRNA (**). [B] Anti-GFP based Western-Blot analyses of whole embryo extracts (~1,7 embryos/lane), microsome preparation (~7,5 embryos/lane) and nuclear extracts (~1,5 embryos/lane). [C] Diagram showing indicated relative signal ratios shown in [A].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664474&req=5

pone.0143825.g006: Rbp7a-GFP and Nmnat1-Rbp7a-GFP are enriched in microsome preparation.[A] Experimental overview indicating the encoded proteins, concentrations of injected mRNA (2nl/embryo) and data quantification of Western-Blot shown in [B]. Relative signal intensities always relate to H2B-GFP signals (*). Signal ratios correspond to the indicated quotient of t relative signal intensities. All mRNA were co-injected with 50ng/μl nucRFP mRNA (**). [B] Anti-GFP based Western-Blot analyses of whole embryo extracts (~1,7 embryos/lane), microsome preparation (~7,5 embryos/lane) and nuclear extracts (~1,5 embryos/lane). [C] Diagram showing indicated relative signal ratios shown in [A].
Mentions: The mild increase in overall NAD+ levels seen in nmnat1-rbp7a injected embryos is unlikely to have a major impact on retinoid metabolism. However, there is evidence for organelle specific, non-redundant activities of Nmnat proteins [42–45]. In humans, the three NMNAT proteins have been shown to exert slightly different enzymatic activities at different subcellular localization [42, 43]. In particular, the use of tagged proteins revealed organelle specific targeting of Nmnat1 to the nucleus, of Nmnat2 to the Golgi complex and of Nmnat3 to the mitochondria. Therefore, we hypothesized that Nmnat1 and Nmnat1-Rbp7a also might differ in their sub-cellular localizations. This notion was supported by protein sequence based prediction analysis (PSORT server) that suggested a nuclear bias for Nmnat1 (56,5%) and a dominant cytoplasmic bias for Nmnat1-Rbp7a (60,9%) and Rbp7a (52,2%, Fig 5A). To verify these predictions, fusion constructs of full-length proteins tagged with C-terminal eGFP were generated and expressed in HEK cells (Fig 5B) as well as in zebrafish embryos (Fig 5C). For the zebrafish experiments, embryos at the 1–2 cell stage were injected with a mixture of two mRNAs, one encoding the GFP-tagged protein and the second encoding a nuclear specific RFP-variant (H2B-RFP) as an injection control. As described for the human fusion protein, zebrafish Nmnat1-GFP was strictly nuclear in both systems (Fig 5B and 5C compare with H2B-GFP). In contrast, Rbp7a-GFP showed the predicted cytoplasmic distribution in HEK cells and an additional slight nuclear enrichment in the early zebrafish embryo. Importantly, also the Nmnat1-Rbp7a-GFP fusion isoform was detected in both the nucleus and the cytoplasm. The data suggest an Rbp7a-dependend expansion of the NAD+ catalyzing activity into the cytoplasm. As the majority of cellular retinal-generating capacity resides in the ER, we speculated that Nmnat1-Rbp7a might function as a ER-specific NAD+ source [8]. To test this option, different cellular fractions prepared from embryos injected with mRNA encoding the different GFP-tagged proteins were analyzed by Western-blots using an GFP-antibody (summarized in Fig 6A, for details see Material and Methods). Importantly, signals for Rbp7a-GFP (lane 3) and Nmnat1-Rbp7a-GFP (lane 5) as compared to Nmnat (lane 4) were much stronger in the ER-containing microsome fractions as compared to whole embryo extracts and the nuclear/debris fractions (termed ‘nuclear fraction’, Fig 6B). Signal quantification revealed that in whole embryo extract the protein levels for Rbp7a-GFP and Nmnat1-Rbp7a-GFP were similar to that of H2B-GFP and roughly 6–8 times lower than those of Nmnat1-GFP (Fig 6A and 6B). In contrast, in the microsome preparation Rbp7a-GFP and Nmnat1-Rbp7a-GFP level were more than 8 times higher than that of H2B-GFP and only 1,5–1,6 lower than that of Nmnat1-GFP. The data correspond to a microsome specific enrichment of Rbp7a-GFP and Nmnat1-Rbp7a-GFP over H2B-GFP by factors of 8,6 and 6,2, respectively (Fig 6A and 6C). This suggests a similar association of Rbp7a and Nmnat1-Rbp7a with microsomal structures, presumably with the ER.

Bottom Line: We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production.Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization.HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology/CMBI, University of Innsbruck, Technikerstrasse 25, 6020, Innsbruck, Austria.

ABSTRACT
Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

Show MeSH